Volume 40, Issue 4, Pages 569-581 (April 2014) Treg Cells Expressing the Coinhibitory Molecule TIGIT Selectively Inhibit Proinflammatory Th1 and Th17 Cell Responses  Nicole Joller, Ester Lozano, Patrick R. Burkett, Bonny Patel, Sheng Xiao, Chen Zhu, Junrong Xia, Tze G. Tan, Esen Sefik, Vijay Yajnik, Arlene H. Sharpe, Francisco J. Quintana, Diane Mathis, Christophe Benoist, David A. Hafler, Vijay K. Kuchroo  Immunity  Volume 40, Issue 4, Pages 569-581 (April 2014) DOI: 10.1016/j.immuni.2014.02.012 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 TIGIT Is Expressed on Highly Suppressive Treg Cells and Promotes iTreg Cell Differentiation (A) CD4+ T cells were purified from Foxp3-GFP.KI mice and the Foxp3+ and Foxp3− cells were sorted. Foxp3+ nTreg cells were stained directly for TIGIT (solid line) or with an isotype control (dotted line) and analyzed by flow cytometry. Foxp3+ induced Treg (iTreg) cells were analyzed after 4 days of stimulation with TGF-β. (B and C) Naive CD4+CD62L+ T cells were sorted from WT, Tigit−/− (B), or Tigit tg (C) mice and differentiated as in (A). Foxp3 expression was analyzed by flow cytometry (mean ± SD). (D) CD4+Foxp3+TIGIT+ (closed diamond) or CD4+Foxp3+TIGIT− (open diamond) Treg cells were sorted from Foxp3-GFP.KI mice and titrated onto Foxp3−GFP− effector T cells stimulated with anti-CD3 and APCs. Proliferation was measured after 72 hr by 3H-thymidine incorporation. Mean ± SD; ∗p < 0.01; representative experiment of >10 independent experiments. (E) Sorting strategy of ex vivo FACS-sorted human effector T cells (CD4+CD25+CD127+) and Treg cells (CD4+CD25hiCD127−) sorted into TIGIT+ and TIGIT−. (F) Treg cells sorted as outlined in (E) showed >96% purity in both subsets measured by Foxp3 staining after isolation. (G) Representative suppression assay with human CD4+CD25hiCD127−TIGIT+ and TIGIT− Treg cells cocultured with CFSE-labeled CD25-depleted CD4+ effector T cells for 4 days. (H) Statistical summary of (G) of six healthy donors (mean ± SEM; ∗p < 0.05). Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Expression Profiling of TIGIT+ Treg Cells (A and B) Heatmap of chemokine (receptor) and cytokine (receptor) (A) or transcription factor (B) genes that are differentially expressed (>1.5-fold) in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Treg cells (duplicate samples are shown). (C) Differential expression of a selection of genes from (B) was determined by quantification of mRNA in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Treg cells by RT-PCR. Mean ± SD of at least three independent experiments is shown. (D–F) Volcano plots comparing the p value versus fold-change for probes from TIGIT+ versus TIGIT− Treg cells. Treg cell signatures generated from (D) CXCR3+ versus CXCR3− Treg cells, (E) WT versus IRF4 KO Treg cells, and (F) Treg cells from GFP-Foxp3 fusion protein reporter mice versus Foxp3-IRES-GFP mice are highlighted in red (overexpressed) and green (underrepresented). p values form a chi-square test. Genes and Probe IDs included in the signatures are listed in Table S2. Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 TIGIT+ Treg Cells Express a Gene Profile Indicative of a Highly Suppressive Treg Cell Subset (A) Heat map of surface receptor genes that are differentially expressed (>1.5-fold, duplicate samples) in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Treg cells. (B and C) Quantitative RT-PCR (B, mean ± SEM) and flow cytometric (C) confirmation for a selection of genes from (A) and (D). (D) Heatmap of differentially expressed genes involved in Treg cell differentiation and function. (E) Volcano plot comparing the p value versus fold-change for probes from TIGIT+ versus TIGIT− Treg cells. The canonical Treg cell signature is highlighted in red (transcripts upregulated in Treg cells) and green (transcripts downregulated in Treg cells). (F) Foxp3 protein expression was quantified by flow cytometry in mouse Foxp3− (Teff) or Foxp3+ (Treg) and human memory T cells (CD4+CD127+CD25med; Teff) and Treg cells (CD4+CD127loCD25hi) (n = 9; ∗p < 0.05). (G) Relative expression of the indicated genes in CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Treg cells was determined by quantitative PCR (mean ± SEM). Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 TIGIT Ligation Induces Fgl2 Expression (A) Foxp3− (Teff) and Foxp3+ (Treg) cells were sorted from Foxp3-GFP.KI mice, stimulated with anti-CD3 and anti-CD28 in the presence of agonistic TIGIT Ab. After 3 days RNA was extracted and Fgl2 and Il10 mRNA was quantified by quantitative RT-PCR. (B) Ex vivo human memory T cells (CD4+CD127+CD25med) and Treg cells (CD4+CD127loCD25hi) were sorted gating into TIGIT+ and TIGIT−. After isolation, cells were cultured in the presence of agonistic anti-TIGIT or isotype control for 4 days. FGL2 expression was quantified by RT-PCR (n = 6; ∗p < 0.05). (C) Mice were immunized s.c. with MOG35–55 peptide in CFA and treated with anti-TIGIT or isotype control antibody. On day 10 cells were restimulated with MOG35-55 peptide for 48 hr. Fgl2 concentrations in the supernatants were determined by ELISA. (D and E) CD4+CD25+TIGIT+ (closed bars) or CD4+CD25+TIGIT− (open bars) Treg cells were sorted from WT, IL-10-deficient (D), or Fgl2-deficent (E) mice and cocultured with CD25− effector T cells stimulated with anti-CD3 and APCs at a ratio of 1:8. Where indicated neutralizing anti-IL-10 (D) or anti-Fgl2 (E) Ab or the respective isotype control Ab was added to the culture. Proliferation was measured after 72 hr by 3H-thymidine incorporation. (F) CD4+Foxp3+TIGIT+ and CD4+Foxp3+TIGIT− Treg cells were sorted from Foxp3-GFP.KI mice and mRNA for Cebpa was measured by RT-PCR. (G) Cells were isolated and stimulated as in (A) and on day 3 Cebpa mRNA was examined by quantitative RT-PCR. (H) ChIP assays were performed on P815 cells expressing TIGIT with anti-CEBPα antibody or an isotype control. The precipitated chromatin was analyzed by quantitative PCR with primers specific for three promoter and four intragenic regions of the Fgl2 gene with predicted CEBPα binding sites. Signals are displayed as percent of the total input chromatin. (I) CD4+Foxp3+ Treg cells were sorted from Foxp3-GFP.KI mice and transfected with a CEBPα overexpression construct (CEBPα) or the empty vector as control (control) and stimulated with anti-CD3/CD28 Dynabeads. Relative expression of Fgl2 mRNA was determined by RT-PCR 4 days later. All panels represent mean ± SD. Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 TIGIT+ Treg Cells Suppress Th1 and Th17 Cell but Not Th2 Cell Responses (A) Naive effector T cells, WT Foxp3+TIGIT− Treg cells, WT Foxp3+TIGIT+ Treg cells, and Fgl2−/− Foxp3+TIGIT+ Treg cells were sorted and cocultured at a ratio of 1:10 under Th1, Th2, or Th17 cell polarizing conditions. After 3 days mRNA was measured by quantitative RT-PCR. On day 5 intracellular cytokines in CD45.1+ T effector cells were determined by flow cytometry (values normalized to unsuppressed controls, mean ± SEM; ∗p < 0.05, ∗∗p < 0.001, paired Student’s t test). (B) Human TIGIT+ and TIGIT− Treg cells (CD4+CD25hiCD127−) were sorted and cocultured with CFSE-labeled CD25-depleted CD4+ T effector cells. Gene expression (qRT-PCR) and intracellular cytokines (flow cytometry) were measured on day 4 (mean ± SEM; n = 6). (C–F) CD25− effector OT-II cells and CD25hi OT-II Treg cells (TIGIT−, TIGIT+, or no Treg cell control) were transferred i.v. into WT recipients and mice were immunized with OVA in CFA. Data shown as mean ± SEM. (C and D) Expansion of Vβ5+ OT-II T cells (C) and proliferation in response to OVA323-339 (D) were determined 10 days later. (E and F) Intracellular cytokine levels were determined by flow cytometry (E) and cytokine concentration in the culture supernatants was determined by cytometric bead array (F). (G–I) CD25− effector OT-II cells and CD25hi OT-II Treg cells (TIGIT−, TIGIT+, or no Treg cell control) were transferred i.v. into WT recipients. Mice were then sensitized with OVA (i.p.) on days 0 and 7 and challenged with aerosolized OVA on days 14–17 to induce allergic airway inflammation. (G) Total numbers of Vβ5+ OT-II cells in lungs, (H) intracellular cytokine levels from lung-infiltrating CD4+ T cells, and (I) total eosinophil numbers in bronchoalveolar lavage fluid were determined by flow cytometry. Pooled data from two experiments are shown (mean ± SEM; n = 8). Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 TIGIT+ Treg Cells Suppress Proinflammatory Responses In Vivo To induce colitis CD45RBhi effector T cells (CD45.1) were transferred into Rag1−/− mice together with TIGIT+ or TIGIT− Treg cells (CD45.2) or no Treg cells as controls (Teff:Treg cell ratio was 4.4:1 for TIGIT+ Treg cells and 3.6:1 for TIGIT− Treg cells). (A and B) Mice were monitored for weight loss over 10 weeks and (B) total colitis scores were determined by histopathology. (C–E) At 10 weeks after transfer, mesenteric LNs were harvested and (C) total number of infiltrating CD4+ T cells, (D) proportion of Foxp3+ Treg cells among CD4+ T cells, and (E) Foxp3 expression among the transferred Treg cell population (CD45.2+) were determined by flow cytometry. (F) Mesenteric LN cells were restimulated in vitro with 0.5 μg/ml anti-CD3 for 3 days and cytokine secretion was determined by cytometric bead array in supernatants. (G and H) CD45RBhi effector T cells (CD45.1) and WT or Fgl2−/− TIGIT+ or TIGIT− Treg cells (CD45.2) were cotransferred into Rag1−/− mice on day −1, which were then immunized with OVA in alum on day 0 and 7. On day 14 (G) total number of CD4+CD45.1+ effector T cells and (H) frequencies of cytokine producing cells upon restimulated with PMA+ionomycin were determined by flow cytometry. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005; all panels show mean ± SD. Immunity 2014 40, 569-581DOI: (10.1016/j.immuni.2014.02.012) Copyright © 2014 Elsevier Inc. Terms and Conditions